Microorganisms having putrescine productivity and process for producing putrescine using the same

ABSTRACT

The present invention relates to a recombinant microorganism capable of producing putrescine, in which the microorganism is modified to have enhanced NCgl2522 activity, thereby producing putrescine in a high yield, and a method for producing putrescine using the microorganism.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. national phase application of InternationalPCT Patent Application No. PCT/KR2014/001509, which was filed on Feb.25, 2014, which claims priority to Korean Patent Application Nos.10-2014-0017243, filed Feb. 14, 2014 and 10-2013-0030020, filed Mar. 20,2013. These applications are incorporated herein by reference in theirentireties.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is HANO 035 01US ST25.txt. The text file is 48 KB,was created on Sep. 21, 2016, and is being submitted electronically viaEFS-Web.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a recombinant microorganism havingimproved putrescine productivity and a method for producing putrescineat a high yield using the same.

2. Description of the Related Art

Polyamines such as spermidine, spermine or the like are present in mostliving cells, and putrescine (or 1,4-butanediamine) is used as aprecursor in spermidine and spermine metabolisms. Putrescine is found inGram-negative bacteria or fungus, and it is present in highconcentrations in various species, suggesting that it has an importantrole in the metabolic pathways of microorganisms.

In general, putrescine is an important raw material in a synthesis ofpolyamine nylon-4, 6 which is produced by reacting with adipic acid.Putrescine is produced mainly by chemical synthesis throughacrylonitrile and succinonitrile from propylene. This chemical synthesisis a three-step process including a catalytic oxidation reaction, areaction using a cyanide compound, and a hydrogenation reaction usinghigh-pressure hydrogen. There are problems in that this chemicalsynthesis is not environment friendly and also consumes a lot of energyleading to depletion of petroleum. Therefore, a more environmentfriendly and energy-effective method involving biomass utilization needsto be developed for putrescine production.

In microorganisms, a biosynthetic pathway of putrescine is the same asroute of arginine synthesis from glutamate to ornithine synthesis.Putrescine can be biosynthesized through two pathways frommicroorganisms. In one pathway, ornithine as an intermediate isdecarboxylated to synthesize putrescine. In the other pathway, agmatineis produced by decarboxylation arginine synthesized from ornithine, andthen putrescine is synthesized from the agmatine (Morris et al., J Biol.Chem. 241: 13, 3129-3135, 1996). These two pathways produce the energyrequired for metabolism or allow the cell to have resistance tooxidative stress.

As a method for producing putrescine using a microorganism, a method forproducing putrescine at a high concentration by transformation of E.coli and Corynebacterium has been reported (International PatentPublication No. WO06/005603; International Patent Publication No.WO09/125924; Qian ZD et al., Biotechnol. Bioeng. 104: 4, 651-662, 2009;Schneider et al., Appl. Microbiol. Biotechnol. 88: 4, 859-868, 2010;Schneider et al., Appl. Microbiol. Biotechnol. 91: 17-30, 2011). Forexample, WO09/125924 discloses a method for producing putrescine in ahigh yield by enhancing ornithine biosynthetic pathway, instead ofinactivating pathways involved in degradation and utilization ofputrescine which are present in E. coli and inactivating conversion ofornithine as a precursor of putrescine to arginine. In addition,Schneider (2010) discloses a method for producing putrescine at a highconcentration by introducing and enhancing a protein capable ofconverting ornithine to putrescine into a Corynebacterium sp. strainhaving no putrescine productivity.

Furthermore, studies on putrescine transporters in E. coli, yeast, plantand animal cells have been actively conducted (K Igarashi, PlantPhysiol. Biochem. 48: 506-512, 2010). Putrescine uptake of E. colioccurs via 4 pathways; potABCD or potFGHI driven by ATP hydrolysis,andpotE as H+ symporter and puuP of the puu pathway. With regard to Kmvalues of these complexes involved in putrescine uptake, those ofPotFGHI, potABCD, potE and puuP are 0.5 mM, 1.5 mM, 1.8 mM, and 3.7 mM,respectively. Among the four putrescine uptake pathways, potFGHI complexis considered as the most suitable. In addition, potE transporter hasboth functions of uptake and excretion of putrescine. Putrescine isimported together with proton into cells at neural pH. However, asputrescine synthase (speF) is expressed under acidic pH conditions,intracellular uptake of extracellular ornithine and extracellularexcretion of putrescine synthesized within cells occur at the same time(Kurihara et. al., J. Bacteriology 191: 8, 2776-2782, 2009).

The known putrescine exporters in yeast are TPO1 and TPO4. These aminoacid sequence are very similar to the amino acid sequence of bacillusmultidrug transporter Blt.

These two exporters share characteristics with potE in E. coli, and theyhave functions of importing putrescine, spermidine, and spermine underbasic conditions and exporting them under acidic conditions. Inaddition, yeast cell over-expressing TPO5 gene is resistant to 120 mMputrescine whereas a mutant disrupted TPO5 gene is sensitive to 90 mMputrescine (Tachihara et. al., J. Biological Chemistry, 280(13):12637-12642, 2005).

Synthesis and degradation, and uptake and excretion of putrescine inanimal cells are regulated in various ways. Although studies onpolyamine excretion have not been done in animal cells as well as in E.coli or yeast, there is a report that an SLC3A2 (arginine/diamineexporter) functions to import arginine into cells and to exportputrescine, acetyl spermidine, and acetyl spermine in colon epithelialcells. However, there has been no report about uptake and export ofputrescine in plant cells (Igarashi et al., Plant Physiol. & Biochem.48: 506-512, 2010).

On the other hand, since Corynebacterium sp. microorganism has noputrescine biosynthetic pathway, studies regarding putrescine exporthave not been studied. According to a recent report, cell growth isrestored and cadaverine productivity is increased by overexpression of acg2983 membrane protein in a strain producing a cadaverine (Kind et.al., Metabolic Engineering 13: 617-627, 2011).

However, there have been no reports about association between putrescineexporter and putrescine productivity or growth of microorganismsproducing putrescine. In the above literature, there is no mention aboutassociation between cg2983 membrane protein and the exporting ability ofputrescine.

In this background, the present inventors have made many efforts todevelop a strain capable of producing putrescine in a higher yield. As aresult, NCgl2522 functions is revealed as a putrescine exporter in aputrescine-producing strain, Corynebacterium sp. microorganism, andputrescine can be produced in a high yield by enhancing NCgl2522activity, compared to the endogenous activity thereof. In addition, theamount of putrescine in a culture medium can be increased by expressingNCgl2522 in E. coli having the putrescine synthetic pathway, and thusthe present inventors suggested that NCgl2522 also functions as aputrescine exporter in E. coli, thereby completing the presentinvention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a recombinantmicroorganism which is modified to have enhanced NCgl2522 activity,thereby produced putrescine in a high yield.

Another object of the present invention is to provide a method forproducing putrescine in a high yield using the microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing that NCgl2522 is included in a clone (B19)finally selected from the transformed colonies introduced withCorynebacterium chromosome library according to the present invention;and

FIG. 2 is the result of evaluating putrescine resistance of theNCgl2522-deleted or -enhanced recombinant strain according to thepresent invention.

1: KCCM11240P

2: KCCM11240P ΔNCgl2522

3: KCCM11240P P(CJ7)-NCgl2522

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one aspect to achieve the above object, the present inventionprovides a microorganism having putrescine productivity, which ismodified to enhance activity of a protein having an amino acid sequencerepresented by SEQ ID NO: 21 or 23.

In one specific embodiment, the present invention provides amicroorganism having putrescine productivity, in which the microorganismis further modified to have weakened activities of ornithinecarbamoyltransferase (ArgF) and a protein (NCgl1221) involved inglutamate export, compared to the endogenous activities thereof, and isintroduced with ornithine decarboxylase (ODC) activity.

In another specific embodiment, the present invention provides amicroorganism having putrescine productivity, in which the ornithinecarbamoyltransferase (ArgF) has an amino acid sequence represented bySEQ ID NO: 29, the protein (NCgl1221) involved in glutamate export hasan amino acid sequence represented by SEQ ID NO: 30, and the ornithinedecarboxylase (ODC) has an amino acid sequence represented by SEQ ID NO:33.

In still another specific embodiment, the present invention provides amicroorganism having putrescine productivity, in which the microorganismis further modified to have enhanced activities ofacetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamatesynthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase(ArgB), and acetylornithine aminotransferase (ArgD), compared to theendogenous activities thereof.

In still another specific embodiment, the present invention provides amicroorganism having putrescine productivity, in which theacetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamatesynthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase(ArgB), and acetylornithine aminotransferase (ArgD) have amino acidsequences represented by SEQ ID NOs: 25, 26, 27 and 28, respectively.

In still another specific embodiment, the present invention provides amicroorganism having putrescine productivity, in which acetyltransferase(NCgl1469) activity of the microorganism is further weakened.

In still another specific embodiment, the present invention provides amicroorganism having putrescine productivity, in which theacetyltransferase has an amino acid sequence represented by SEQ ID NO:31 or 32.

In still another specific embodiment, the present invention provides amicroorganism having putrescine productivity, in which the microorganismis an Escherichia sp. or a Corynebacterium sp.

In still another specific embodiment, the present invention provides amicroorganism having putrescine productivity, in which the microorganismis E. coli or Corynebacterium glutamicum.

In another aspect, the present invention provides a method for producingputrescine, comprising the steps of culturing a microorganism havingputrescine productivity to obtain a cell culture and recoveringputrescine from the cultured microorganism or cell culture.

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant Corynebacterium sp.microorganism, in which the Corynebacterium sp. microorganism havingputrescine productivity is modified to have enhanced NCgl2522 activity,compared to the endogenous activity thereof and thus it has improvedputrescine productivity.

As used herein, the term “NCgl2522” refers to permease belonging to MFS(major facilitator superfamily), which is a membrane protein isolatedfrom Corynebacterium glutamicum ATCC13032. NCgl2522 is known to exportdiaminopentane from Corynebacterium glutamicum. In the presentinvention, NCgl2522 was confirmed to function as a transporter thatserves to extracellularly export putrescine produced within cells. Onthe basis of this fact, the present invention provides a recombinantmicroorganism showing high-yield putrescine productivity, in whichNCgl2522 is modified to have enhanced activity, compared to theendogenous activity thereof, and therefore, export of intracellularlyproduced putrescine is increased.

As used herein, the term “endogenous activity” refers to the activity ofan enzyme that a microorganism possesses in its native state, namely inthe state without modification, and the meaning of “modified to haveenhanced activity, compared to the endogenous activity” is that theactivity of the enzyme is newly introduced or further improved, comparedto the activity of the corresponding enzyme before modification.

In the present invention, “enhancement of enzymatic activity” includesimprovement in the enzymatic activity by improvement in endogenous geneactivity, amplification of the endogenous gene by internal or externalfactors, deletion of a regulatory factor for suppressing the geneexpression, increase in the gene copy number, increase in the activityby introduction of a foreign gene or modification of an expressionregulatory sequence, in particular, replacement or modification of apromoter and mutation within gene, as well as introduction orimprovement of the activity of the enzyme itself to achieve effectsbeyond the endogenous functions.

In the present invention, “modified to have enhanced activity, comparedto the endogenous activity” means that the activity of the microorganismis increased after manipulation such as introduction of a gene showingthe activity, or increase in the gene copy number, deletion of aregulatory factor for suppressing the gene expression or modification ofan expression regulatory sequence, for example, use of an improvedpromoter, compared to the activity of the microorganism before themanipulation.

The NCgl2522, having its activity is increased by the present invention,may be, but is not particularly limited to, a protein having an aminoacid sequence of SEQ ID NO: 21 or 23 or an amino acid sequence having70% or more homology thereto, preferably 80% or more homology thereto,more preferably 90% or more homology thereto, much more preferably 95%or more homology thereto, much more preferably 98% or more homologythereto, and most preferably 99% or more homology thereto. Further,because the amino acid sequence of the protein showing the activity maydiffer depending on species or strain of the microorganism, the proteinis not limited thereto. That is, the protein may be a protein mutant oran artificial variant that has an amino acid sequence includingsubstitution, deletion, insertion, or addition of one or several aminoacids at one or more positions of the amino acid sequence of SEQ ID NO:21 or 23, as long as the protein aids to improve putrescine productivityby enhancing its activity. As used herein, the term “several” aminoacids means specifically 2 to 20, preferably 2 to 10, and morepreferably 2 to 5 amino acids, although it may differ depending on theposition or type of amino acid residue in the three-dimensionalstructure of the protein. Furthermore, the substitution, deletion,insertion, addition or inversion of amino acids may include naturallyoccurring mutations which occur due to differences of individual orspecies of the microorganism having the activity of the polypeptide orartificial variation.

There are no putrescine biosynthetic pathways in Corynebacterium sp.microorganism. However, when external ornithine decarboxylase (ODC) isintroduced, putrescine is synthesized and excreted extracellularly,indicating presence of a transporter, that is, an exporter thatfunctions as a passage of putrescine among numerous membrane proteins ofCorynebacterium sp. microorganism. Accordingly, in order to isolate theputrescine exporter in Corynebacterium sp. microorganism, the presentinventors prepared a chromosome library of the wild-type Corynebacteriumglutamicum ATCC13032, and they transformed a putrescine-producingstrain, Corynebacterium glutamicum KCCM11138P with the library, andselected strains that grow in a minimal medium containing putrescine.Through tertiary colony selection, a clone (B19) having putrescineresistance was finally selected and base sequence analysis was performedto confirm that the clone contains NCgl2522 (see FIG. 1). As theputrescine exporter, NCgl2522 derived from Corynebacterium glutamicumATCC13032 has the amino acid sequence represented by SEQ ID NO: 21, andNCgl2522 derived from Corynebacterium glutamicum ATCC13869 which has 98%homology to the above amino acid sequence has the amino acid sequencerepresented by SEQ ID NO: 23.

A polynucleotide encoding NCgl2522 of the present invention may includea polynucleotide encoding the protein having the amino acid sequence ofSEQ ID NO: 21 or 23, or the amino acid sequence having 70% or morehomology thereto, preferably 80% or more homology thereto, morepreferably 90% or more homology thereto, much more preferably 95% ormore homology thereto, much more preferably 98% or more homologythereto, and most preferably 99% or more homology thereto, as long asthe protein has the activity similar to that of the NCgl2522 protein,and most preferably, it may include a nucleotide sequence of SEQ ID NO:20 or 22.

As used herein, the term “homology” refers to the similarity between twoamino acid sequences, and can be determined using the well-known methodsusing BLAST 2.0, which calculates parameters such as score, identity,and similarity.

Further, the polynucleotide encoding NCgl2522 of the present inventionmay be a variant which hybridizes under stringent conditions with thenucleotide sequence of SEQ ID NO: 20 or 22, or a probe derived from theabove nucleotide sequence, provided that it encodes a functionalNCgl2522. As used herein, the term “stringent conditions” meanconditions allowing a specific hybridization between polynucleotides.For example, such stringent conditions are described in detail in theliterature (J. Sambrook et al., Molecular Cloning, A Laboratory Manual,2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor,N.Y., 1989; F. M. Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, Inc., New York).

In the present invention, “modified to have enhanced NCgl2522 activity,compared to the endogenous activity” may be performed by a methodselected from methods of increasing the copy number of thepolynucleotide encoding the protein, modifying an expression regulatorysequence to increase expression of the polynucleotide, modifying thepolynucleotide sequence on the chromosome to enhance the activity of theenzyme, deleting a regulatory factor for suppressing the geneexpression, and combinations thereof.

The copy number of the polynucleotide may be, but is not particularlylimited to, increased by operably linking the polynucleotide to a vectoror by integrating it into the host cell genome. Specifically, the copynumber of the polynucleotide in the host cell genome can be increased byintroducing into the host cell the vector which is operably linked tothe polynucleotide encoding the protein of the present invention andreplicates and functions independently of the host cell, or byintroducing into the host cell the vector which is operably linked tothe polynucleotide and is able to integrate the polynucleotide into thehost cell genome.

As used herein, the term “vector” refers to a DNA construct including anucleotide sequence encoding the desired protein, which is operablylinked to an appropriate expression regulatory sequence to express thedesired protein in a suitable host cell. The regulatory sequenceincludes a promoter that can initiate transcription, an optionaloperator sequence for regulating the transcription, a sequence encodinga suitable mRNA ribosome binding site, and a sequence regulating thetermination of transcription and translation. After the vector istransformed into the suitable host cell, it can replicate or functionindependently of the host genome, and can be integrated into the genomeitself.

The vector used in the present invention is not particularly limited, aslong as it is able to replicate in the host cell, and any vector knownin the art can be used. Examples of conventional vectors may include anatural or recombinant plasmid, cosmid, virus and bacteriophage. Forinstance, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A,and Charon21A may be used as a phage vector or cosmid vector. As aplasmid vector, pBR type, pUC type, pBluescriptII type, pGEM type, pTZtype, pCL type and pET type may be used. A vector usable in the presentinvention is not particularly limited, and any known expression vectorcan be used. Preferably, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19,pBR322, pMW118, or pCC1BAC vector may be used, and more preferably, pDZvector may be used.

Further, the polynucleotide encoding the desired protein in thechromosome can be replaced by a mutated polynucleotide using a vectorfor chromosomal insertion. The insertion of the polynucleotide into thechromosome may be performed by any method known in the art, for example,homologous recombination. Since the vector of the present invention canbe inserted into the chromosome by homologous recombination, it mayfurther include a selection marker to confirm chromosomal insertion. Theselection marker is to select cells transformed with the vector, thatis, to confirm insertion of the desired polynucleotide, and theselection marker may include markers providing selectable phenotypes,such as drug resistance, auxotrophy, resistance to cytotoxic agents, orsurface protein expression. Only cells expressing the selection markerare able to survive or to show different phenotypes under theenvironment treated with the selective agent, and thus the transformedcells can be selected.

As used herein, the term “transformation” means the introduction of avector including a polynucleotide encoding a target protein into a hostcell in such a way that the protein encoded by the polynucleotide isexpressed in the host cell. As long as the transformed polynucleotidecan be expressed in the host cell, it can be either integrated into orplaced in the chromosome of the host cell, or exist extrachromosomally.Further, the polynucleotide includes DNA and RNA encoding the targetprotein. The polynucleotide can be introduced in any form, as long as itcan be introduced into the host cell and expressed therein. For example,the polynucleotide can be introduced into the host cell in the form ofan expression cassette, which is a gene construct including all elementsrequired for its autonomous expression. Typically, the expressioncassette includes a promoter operably linked to the polynucleotide,transcriptional termination signals, ribosome binding sites, ortranslation termination signals. The expression cassette may be in theform of a self-replicable expression vector. Also, the polynucleotide asit is may be introduced into the host cell and operably linked tosequences required for expression in the host cell.

Further, as used herein, the term “operably linked” means a functionallinkage between a polynucleotide sequence encoding the desired proteinand a promoter sequence which initiates and mediates transcription ofthe polynucleotide sequence.

As well, modification of the expression regulatory sequence forincreasing the polynucleotide expression may be, but is not limited to,done by inducing a modification on the expression regulatory sequencethrough deletion, insertion, non-conservative or conservativesubstitution of nucleotide sequence, or a combination thereof in orderto further enhance the activity of expression regulatory sequence, or byreplacing the expression regulatory sequence with a nucleotide sequencehaving stronger activity. The expression regulatory sequence includes,but is not particularly limited to, a promoter, an operator sequence, asequence coding for ribosome-binding site, and a sequence regulating thetermination of transcription and translation.

A strong heterologous promoter instead of the original promoter may belinked upstream of the polynucleotide expression unit, and examples ofthe strong promoter may include CJ7 promoter, lysCP1 promoter, EF-Tupromoter, groEL promoter, aceA or aceB promoter, and more preferably,lysCP1 promoter or CJ7 promoter as a Corynebacterium-derived promoter,and the polynucleotide encoding the enzyme is operably linked thereto sothat its expression rate can be increased. Herein, the lysCP1 promoteris a promoter improved through nucleotide sequence substitution of thepromoter region of the polynucleotide encoding aspartate kinase andaspartate semialdehyde dehydrogenase, and is a strong promoter thatincreases expression of the aspartate kinase gene, leading to 5-foldincreased activity of the corresponding enzyme, compared to thewild-type (WO 2009/096689). Further, CJ7 promoter is a promoter that wasfound during exploration of a strong promoter sequence inCorynebacterium ammoniagenes and confirmed to be expressed inCorynebacterium ammoniagenes and Escherichia and to have a strongpromoter activity. CJ7 promoter is a promoter that also shows highexpression activity in Corynebacterium glutamicum (Korean Patent No.0620092 and WO 2006/065095).

Furthermore, modification of a polynucleotide sequence on chromosome maybe, but is not particularly limited to, done by inducing a mutation onthe expression regulatory sequence through deletion, insertion,non-conservative or conservative substitution of polynucleotidesequence, or a combination thereof in order to further enhance theactivity of the polynucleotide sequence, or by replacing the sequencewith a polynucleotide sequence which is modified to have strongeractivity.

In one preferred embodiment of the present invention, in order toprovide a Corynebacterium sp. microorganism having improved putrescineproductivity, the copy number of the gene can be increased byintroducing into the chromosome the polynucleotide having the nucleotidesequence of SEQ ID NO: 20 or 22 encoding NCgl2522 involved in putrescineexcretion, or the own promoter of NCgl2522 can be substituted with apromoter having improved activity, preferably, CJ7 promoter having thenucleotide sequence of SEQ ID NO: 24.

As used herein, the term “microorganism having putrescine productivity”or “microorganism producing putrescine” refers to a microorganism thatis prepared by providing putrescine productivity for the parent strainhaving no putrescine productivity. The microorganism that is providedwith putrescine productivity or produces putrescine may be, but is notparticularly limited to, a microorganism having improved productivity ofornithine to be used as a raw material for putrescine biosynthesis, inwhich the microorganism is modified to have higher activities ofacetylglutamate synthase converting glutamate to acetylglutamate(Nacetylglutamate) or ornithine acetyltransferase (ArgJ) convertingacetyl ornithine to ornithine, acetylglutamate kinase (ArgB) convertingacetyl glutamate to acetylglutamyl phosphate (N-acetylglutamylphosphate), acetyl-gamma-glutamyl phosphate reductase (ArgC) convertingacetyl glutamyl phosphate to acetyl glutamate semialdehyde (N-acetylglutamate semialdehyde), or acetylornithine aminotransferase (ArgD)converting acetyl glutamate semialdehyde to acetylornithine(N-acetylornithine) than the endogenous activity, in order to enhancethe biosynthetic pathway from glutamate to ornithine. Further, themicroorganism is a microorganism that is modified to have weakeractivity of ornithine carbamoyltransferase (ArgF) involved in synthesisof arginine from ornithine, the protein (NCgl1221) involved in glutamateexcretion, and/or the protein (NCgl469) acetylating putrescine than theendogenous activity, and/or modified to have ornithine decarboxylase(ODC) activity.

In this regard, acetyl-gamma-glutamyl-phosphate reductase (ArgC),acetylglutamate synthase or ornithineacetyltransferase (ArgJ),acetylglutamate kinase (ArgB), acetylornithine aminotransferase (ArgD),ornithine carbamoyl transferase (ArgF), the protein (NCgl1221) involvedin glutamate export, and ornithine decarboxylase (ODC) may have, but arenot particularly limited to, preferably the amino acid sequencesrepresented by SEQ ID NOs: 25, 26, 27, 28, 29, 30 and 33, respectively,or amino acid sequences having 70% or more homology thereto, morepreferably 80% or more homology thereto, or much more preferably 90% ormore homology thereto, respectively. In addition, the protein (NCgl469)acetylating putrescine may have, but is not particularly limited to,preferably the amino acid sequence represented by SEQ ID NO: 31 or 32,or an amino acid sequence having 70% or more homology thereto, morepreferably 80% or more homology thereto, or much more preferably 90% ormore homology thereto.

Of the proteins, the increase in the activities ofacetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamatesynthase or ornithineacetyltransferase (ArgJ), acetylglutamate kinase(ArgB), acetylornithine aminotransferase (ArgD), and ornithinedecarboxylase (ODC) may be achieved by the above described method ofincreasing the NCgl2522 activity, for example, a method selected fromthe methods of increasing the copy number of the polynucleotide encodingthe protein, modifying an expression regulatory sequence to increaseexpression of the polynucleotide, modifying the polynucleotide sequenceon the chromosome to enhance the activity of the enzyme, deleting aregulatory factor to suppress the expression of the polynucleotide ofthe enzyme, and combinations thereof.

Further, activities of ornithine carbamoyl transferase (ArgF), theprotein (NCgl1221) involved in glutamate export, and the protein(NCgl469) acetylating putrescine can be diminished by a method selectedfrom the group consisting of a partial or full deletion of apolynucleotide encoding the protein, modification of an expressionregulatory sequence for suppressing the polynucleotide expression,modification of the polynucleotide sequence on chromosome fordiminishing the protein activity, and a combination thereof.

In detail, a partial or full deletion of the polynucleotide encoding theprotein can be done by introducing a vector for chromosomal insertioninto a microorganism, thereby substituting the polynucleotide encodingan endogenous target protein on chromosome with a partially removedpolynucleotide or a marker gene. The “partial” may vary depending on thetype of polynucleotide, but specifically refers to 1 to 300, preferably1 to 100, and more preferably 1 to 50 nucleotides.

Also, modification of the expression regulatory sequence can be done byinducing a modification on the expression regulatory sequence throughdeletion, insertion, non-conservative or conservative substitution ofnucleotide sequence, or a combination thereof in order to diminish theactivity of expression regulatory sequence, or by replacing theexpression regulatory sequence with a nucleotide sequence having weakeractivity. The expression regulatory sequence includes a promoter, anoperator sequence, a sequence coding for ribosome-binding site, and asequence regulating the termination of transcription and translation.

Furthermore, modification of a polynucleotide sequence on chromosome canbe done by inducing a mutation on the sequence through deletion,insertion, non-conservative or conservative substitution ofpolynucleotide sequence, or a combination thereof in order to furtherdiminish the enzymatic activity, or by replacing the sequence with apolynucleotide sequence which is modified to have weaker activity.

Moreover, a regulatory factor for suppressing the expression of thepolynucleotide of the enzyme can be deleted by substituting apolynucleotide of the expression suppressing factor with a partiallyremoved polynucleotide or a marker gene. The “partial” may varydepending on the type of polynucleotide, but specifically refers to 1 to300, preferably 1 to 100, and more preferably 1 to 50 nucleotides.

Meanwhile, the microorganism of the present invention is a microorganismhaving putrescine productivity, and includes a prokaryotic microorganismexpressing the protein having the amino acid sequence represented by SEQID NO: 21 or 23, and examples thereof may include microorganismsbelonging to Escherichia sp., Shigella sp., Citrobacter sp., Salmonellasp., Enterobacter sp., Yersinia sp., Klebsiella sp., Erwinia sp.,Corynebacterium sp., Brevibacterium sp., Lactobacillus sp., Selenomanassp., Vibrio sp., Pseudomonas sp., Streptomyces sp., Arcanobacterium sp.,Alcaligenes sp. or the like. The microorganism of the present inventionis preferably a microorganism belonging to Escherichia sp. or amicroorganism belonging to Corynebacterium sp., and more preferably, E.coli or Corynebacterium glutamicum.

In a specific embodiment of the present invention, a Corynebacterium sp.microorganism with Accession No. KCCM11138P (Korean Patent PublicationNO. 2012-0064046) and a Corynebacterium sp. microorganism with AccessionNo. KCCM11240P (Korean Patent Application NO. 2012-0003634) were used asstrains that have enhanced synthetic pathway from glutamate toputrescine, thereby producing putrescine at a high concentration.

In still another embodiment of the present invention, Corynebacteriumglutamicum ATCC13032-based putrescine-producing strains, KCCM11138P andKCCM11240P, and Corynebacterium glutamicum ATCC13869-basedputrescine-producing strains DAB12-a and DAB12-b having the samegenotype were used. ATCC13869 strain can be obtained from American TypeCulture Collection (ATCC). That is, a unique accession number is listedin the catalog of ATCC is given for each strain, and the strain can beordered using the accession number. Specifically, theputrescine-producing strain DAB12-a is characterized by deletion of agene encoding ornithine carbamoyl transferase (ArgF) and a gene encodingthe glutamate exporter NCgl1221, introduction of a gene encodingornithine decarboxylase (OCD), and replacement of the promoter ofornithine biosynthetic gene operon (argCJBD) by an improved promoter inthe Corynebacterium glutamicum ATCC13869. Further, theputrescine-producing strain DAB12-b is characterized in that it isprepared by modifying the DAB12-a strain to have weakened activity ofthe protein (NCgl1469) acetylating putrescine, compared to theendogenous activity.

According to one preferred Example, Corynebacterium glutamicumKCCM11138P prepared by deletion of the gene encoding ornithine carbamoyltransferase (ArgF) and a gene encoding the glutamate exporter NCgl1221,replacement of the own promoter of ArgCJBD gene cluster encoding anenzyme involved in the synthesis of ornithine from glutamate by animproved promoter, and introduction of the gene encoding ornithinedecarboxylase (ODC) into the chromosome in the wild-type Corynebacteriumglutamicum ATCC13032, and Corynebacterium glutamicum KCCM11240P preparedby additionally weakening a gene encoding the acetyltransferase NCgl1469in the microorganism were prepared as putrescine-producing strains.

Meanwhile, in order to prepare an NCgl2522-deleted strain derived fromCorynebacterium glutamicum ATCC13032, a plasmid pDZ-1′NCgl2522(K/O) wasprepared, based on the nucleotide sequence of NCgl2522 derived fromCorynebacterium glutamicum ATCC13032.

The plasmid pDZ-1′NCgl2522(K/O) was transformed into the preparedputrescine-producing strains, KCCM11138P and KCCM11240P, and selected asNCgl2522-deleted strains, and these strains were designated asKCCM11138P ΔNCgl2522 and KCCM11240P ΔNCgl2522, respectively. In the samemanner, NCgl2522-deleted strains derived from Corynebacterium glutamicumATCC13869 were prepared and designated as DAB12-a ΔNCgl2522 and DAB12-bΔNCgl2522.

Putrescine productivities of 4 types of NCgl2522-deleted strains thusprepared were compared with that of the parent strain, and as a result,putrescine productivity was reduced in all of NCgl2522-deletedKCCM11138P ΔNCgl2522, KCCM11240P Δ NCgl2522, DAB12-a Δ NCgl2522, andDAB12-b Δ NCgl2522, compared to the parent strain (see Table 3). Basedon this result, the present inventors confirmed that NCgl2522 activityin the putrescine-producing strain is closely related to putrescineproductivity, and they prepared NCgl2522-enhanced strains in order toincrease putrescine productivity through enhancement of the activity.

To this end, in one preferred Example of the present invention, NCgl2522was additionally introduced into the transposon of Corynebacteriumglutamicum strain or the own NCgl2522 promoter within the chromosome wasreplaced by the CJ7 promoter (KCCM10617, Korean Patent NO. 10-0620092)that was newly developed by the present inventors.

Putrescine productivities of 6 types of NCgl2522-enhanced strains thusprepared were compared with that of the parent strain, and as a result,putrescine productivity was increased in all of the strains prepared byadditional introduction of NCgl2522 into the transposon, compared to theparent strain (see Table 6). Intracellular putrescine concentrationswere measured in the NCgl2522-enhanced strains showing an improvement inputrescine productivity, and as a result, they showed reductions ofintracellular putrescine concentrations, compared to the parent strain(see Table 9). Based on these results, the present inventors confirmedthat extracellular export of putrescine intracellularly produced isincreased by enhancing NCgl2522 activity in the putrescine-producingstrains, thereby improving putrescine productivity.

Accordingly, the Corynebacterium sp. microorganism having an enhancedputrescine productivity, in which the putrescine-producing strainCorynebacterium glutamicum KCCM11138P was modified to have enhancedNCgl2522 activity, compared to the endogenous activity, and thusexhibits an enhanced ability to export putrescine, was designated asCorynebacterium glutamicum CC01-0510, and deposited under the BudapestTreaty to the Korean Culture Center of Microorganisms (KCCM) on Mar. 8,2013, with Accession No. KCCM11401P.

According to another aspect of the present invention, the presentinvention provides a method for producing putrescine, including thesteps of:

(i) culturing the microorganism having putrescine productivity to obtaina cell culture; and

(ii) recovering putrescine from the cultured microorganism or the cellculture.

In the method, the step of culturing the microorganism may be, but isnot particularly limited to, preferably performed by batch culture,continuous culture, and fed-batch culture known in the art. In thisregard, the culture conditions are not particularly limited, but anoptimal pH (e.g., pH 5 to 9, preferably pH 6 to 8, and most preferablypH 6.8) can be maintained by using a basic chemical (e.g., sodiumhydroxide, potassium hydroxide or ammonia) or acidic chemical (e.g.,phosphoric acid or sulfuric acid). Also, an aerobic condition can bemaintained by adding oxygen or oxygen-containing gas mixture to a cellculture. The culture temperature may be maintained at 20 to 45° C., andpreferably at 25 to 40° C. In addition, the cultivation is preferablyperformed for about 10 to 160 hours. The putrescine produced by theabove cultivation may be excreted to a culture medium or remain insidethe cell.

Furthermore, the culture medium to be used may include sugar andcarbohydrate (e.g., glucose, sucrose, lactose, fructose, maltose,molasse, starch and cellulose), oil and fat (e.g., soybean oil,sunflower seed oil, peanut oil and coconut oil), fatty acid (e.g.,palmitic acid, stearic acid and linoleic acid), alcohol (e.g., glyceroland ethanol), and organic acid (e.g., acetic acid) individually or incombination as a carbon source; nitrogen-containing organic compound(e.g., peptone, yeast extract, meat juice, malt extract, corn solution,soybean meal powder and urea), or inorganic compound (e.g., ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, andammonium nitrate) individually or in combination as a nitrogen source;potassium dihydrogen phosphate, dipotassium phosphate, orsodium-containing salt corresponding thereto individually or incombination as a phosphorus source; other essential growth-stimulatingsubstances including metal salts (e.g., magnesium sulfate or ironsulfate), amino acids, and vitamins.

The method for recovering putrescine that is produced in the culturingstep of the present invention can be carried out, for example, using asuitable method known in the art according to batch culture, continuousculture, or fed-batch culture, thereby collecting the desired aminoacids from the culture.

Hereinafter, the present invention will be described in more detail withreference to Examples. However, these Examples are for illustrativepurposes only, and the invention is not intended to be limited by theseExamples.

REFERENCE EXAMPLE 1 Preparation of Corynebacterium sp. MicroorganismHaving Putrescine Productivity

In order to prepare a Corynebacterium sp. microorganism havingputrescine productivity, the biosynthetic pathway of arginine fromornithine was blocked, the biosynthetic pathway of ornithine fromglutamate was enhanced, and the foreign ornithine decarboxylase (OCD)was introduced to prepare a microorganism provided with putrescineproductivity, as described in Korean Patent Publication NO.10-2012-0064046.

Specifically, based on the Corynebacterium glutamicum ATCC13032, a geneencoding ornithine carbamoyltransferase (ArgF) and a gene encodingNCgl1221 which is a protein involved in glutamate export in thechromosome of the strain were deleted by homologous recombination so asto increase the intracellular content of glutamate which is a precursorof ornithine. Further, a gene encoding ornithine decarboxylase (ODC)derived from the wild-type E. coli W3110 which is involved in thesynthesis of putrescine from ornithine was introduced into thechromosome of the strain. Furthermore, the own promoter of argCJBD genecluster which codes for an enzyme involved in the synthesis of ornithinefrom glutamate was replaced by an improved promoter CJ7 promoter toprepare a Corynebacterium glutamicum strain having putrescineproductivity. At this time, argCJBD encodes acetyl gamma glutamylphosphate reductase (ArgC), acetylglutamate synthase or ornithineacetyltransferase (ArgJ), acetylglutamate kinase (ArgB), acetylornithine aminotransferase (ArgD) which are involved in the biosyntheticpathway of ornithine from glutamate. The Corynebacterium glutamicumstrain having putrescine productivity thus prepared was deposited underthe Budapest Treaty to the Korean Culture Center of Microorganisms(KCCM) on Nov. 24, 2010, with Accession No. KCCM11138P. A detaileddescription concerning the preparation of Corynebacterium sp.microorganism having putrescine productivity is given in Korean PatentPublication No. 10-2012-0064046, the disclosure of which is incorporatedby reference is in its entirety.

REFERENCE EXAMPLE 2 Preparation of Corynebacterium sp. MicroorganismHaving Putrescine Productivity

A gene encoding acetyltransferase NCgl1469 in the Corynebacteriumglutamicum KCCM11138P prepared in Reference Example 1 was weakened toproduce no N-acetyl putrescine, thereby Corynebacterium glutamicumstrain having improved putrescine productivity was prepared as anotherCorynebacterium sp. microorganism having putrescine productivity.

Specifically, based on the nucleotide sequence of the gene encodingNCgl1469 of Corynebacterium glutamicum ATCC13032, a pair of primers ofSEQ ID NOs: 1 and 2 for obtaining a homologous recombination fragment ofthe N-terminal region of NCgl1469 and a pair of primers of SEQ ID NOs: 3and 4 for obtaining a homologous recombination fragment of theC-terminal region of NCgl1469 were constructed as in the following Table1.

TABLE 1 Primer Sequence (5′-3′) NCgl1469-del-F1_CGGGATCCAACCTTCAGAACGCGAATAC BamHI (SEQ ID NO: 1) NCgl1469-del-R1_CGCGTCGACTTGGCACTGTGATTACCATC SalI (SEQ ID NO: 2) NCgl1469-del-F2_CGCGTCGACTTGGGTTATATCCCCTCAGA SalI (SEQ ID NO: 3) NCgl1469-del-R2_TGCTCTAGATAGTGAGCCAAGACATGGAA XbaI (SEQ ID NO: 4)

PCR was performed using the genomic DNA of Corynebacterium glutamicumATCC13032 as a template and two pairs of primers so as to obtain PCRfragments of the N-terminal and C-terminal regions, respectively. ThesePCR fragments were electrophoresed to obtain the desired fragments. Atthis time, PCR reaction was carried out for 30 cycle of denaturation for30 seconds at 95° C., annealing for 30 seconds at 55° C., and extensionfor 30 seconds at 72° C. The fragment of the N-terminal region thusobtained was treated with restriction enzymes, BamHI and SalI and thefragment of the C-terminal region thus obtained was treated withrestriction enzymes, SalI and XbaI. The fragments thus treated werecloned into a pDZ vector treated with restriction enzymes, BamHI andXbaI, so as to construct a plasmid pDZ-NCgl1469(K/O).

The plasmid pDZ-NCgl1469(K/O) was transformed into Corynebacteriumglutamicum KCCM11138P by electroporation to obtain a transformant. Then,the transformant was plated and cultured on BHIS plate (37 g/l of Braineheart infusion, 91 g/l of sorbitol, 2% agar) containing kanamycin (25μg/ml) and X-gal (5-bromo-4-chloro-3-indolin-D-galactoside) for colonyformation. From the colonies formed on the plate, blue-colored colonieswere selected as the strain introduced with the plasmidpDZ-NCgl1469(K/O).

The selected strain was inoculated in CM medium (10 g/l of glucose, 10g/l of polypeptone, 5 g/l of yeast extract, 5 g/l of beef extract, 2.5g/l of NaCl, 2 g/l of urea, pH 6.8) and cultured with shaking at 30° C.for 8 hours. Subsequently, each cell culture was serially diluted from10⁻⁴ to 10⁻¹⁰. Then diluted samples were plated and cultured on anX-gal-containing solid medium for colony formation.

From the colonies formed, the white colonies which appear at relativelylow frequency were selected to prepare a Corynebacterium glutamicumstrain having improved putrescine productivity by deletion of the geneencoding NCgl1469. The Corynebacterium glutamicum strain having improvedputrescine productivity thus prepared was designated as KCCM11138PΔNCgl1469 and deposited under the Budapest Treaty to the Korean CultureCenter of Microorganisms (KCCM) on Dec. 26, 2011, with Accession No.KCCM11240P. A detailed description concerning the preparation ofCorynebacterium sp. microorganism having putrescine productivity isgiven in Korean Patent Application No. 10-2012-0003634, the disclosureof which is incorporated by reference is in its entirety.

EXAMPLE 1 Exploration of Putrescine Exporter and Selection of LibraryClones with Putrescine Resistance

Corynebacterium glutamicum has no putrescine biosynthetic pathways.However, when Corynebacterium glutamicum is introduced with externalornithine decarboxylase to have an ability to produce putrescine, itproduces and excretes putrescine extracellularly. It is indicated thepresence of a transporter protein that functions as a passage ofputrescine among numerous membrane proteins of Corynebacterium sp.microorganism.

In order to separate and isolate the putrescine exporter from theCorynebacterium sp. microorganism, a chromosome library of the wild-typeCorynebacterium glutamicum ATCC13032 was prepared. Specifically, thechromosome of the Corynebacterium glutamicum ATCC13032 was treated withthe restriction enzyme Sau3AI for incomplete cleavage. A gene fragmentof 3˜5 kb was separated, and cloned into a pECCG122 vector treated withBamHI (shuttle vector of E. coli and Corynebacterium; Korean PatentPublication No. 10-1992-0000933).

The Corynebacterium chromosome library thus obtained was transformedinto the putrescine-producing strain, Corynebacterium glutamicumKCCM11138P according to Reference Example 1, and then strains growing in0.35 M putrescine-containing minimal medium (containing 10 g of glucose,0.4 g of MgSO₄.7H₂O, 4 g of NH₄Cl, 1 g of KH₂PO₄, 1 g of K₂HPO₄, 2 g ofurea, 10 mg of FeSO₄.7H₂O, 1 mg of MnSO₄.5H₂O, 5 mg of nicotinamide, 5mg of thiamine hydrochloride, 0.1 mg of biotin, 1 mM arginine, 25 mg ofkanamycin, 0.35 M putrescine, based on 1 l of distilled water, pH 7.0)were selected. From about 5.5×10⁵ transformants introduced with theCorynebacterium chromosome library, 413 colonies were selected, and theneach library clone, of which putrescine resistance was also confirmed bysecondary examination, was re-introduced into the putrescine-producingstrain. Finally, one clone (B19), of which putrescine resistance wasconfirmed by tertiary examination, was selected. The clone was subjectedto nucleotide sequence analysis. As a result, It was found to haveNCgl2522 in B19 clone (FIG. 1).

NCgl2522 which was isolated as the putrescine exporter fromCorynebacterium glutamicum ATCC13032 has the amino acid sequencerepresented by SEQ ID NO: 21 which is encoded by a polynucleotide havingthe nucleotide sequence represented by SEQ ID NO: 20.

EXAMPLE 2 Preparation of NCgl2522-deleted Strain and Examination of itsPutrescine Productivity

<2-1> Preparation of NCgl2522-deleted Strain from ATCC13032-basedPutrescine-Producing Strain

In order to examine whether the Corynebacterium glutamicumATCC13032-derived NCgl2522 is involved in putrescine export, a vectorfor deleting the gene encoding NCgl2522 was constructed.

Specifically, based on the nucleotide sequence of the gene encodingNCgl1469 which is represented by SEQ ID NO: 20, a pair of primers of SEQID NOs: 5 and 6 for obtaining a homologous recombination fragment of theN-terminal region of NCgl1469 and a pair of primers of SEQ ID NOs: 7 and8 for obtaining a homologous recombination fragment of the C-terminalregion of NCgl1469 were constructed as in the following Table 2.

TABLE 2 Primer Sequence (5′-3′) NCgl2522-del-F1_BamHICGGGATCCCACGCCTGTCTGGTCGC (SEQ ID NO: 5) NCgl2522-del-R1_SalIACGCGTCGACGGATCGTAACTGTAAC- (SEQ ID NO: 6) GAATGG NCgl2522-del-F2_SalIACGCGTCGACCGCGTGCATCTTT- (SEQ ID NO: 7) GGACAC NCgl2522-del-R2_XbaICTAGTCTAGAGAGCTGCAC- (SEQ ID NO: 8) CAGGTAGACG

PCR was performed using the genomic DNA of Corynebacterium glutamicumATCC13032 as a template and two pairs of primers so as to amplify PCRfragments of the N-terminal and C-terminal regions of NCgl2522 gene.These PCR fragments were electrophoresed to obtain the desiredfragments. At this time, PCR reaction was carried out for 30 cycle ofdenaturation for 30 seconds at 95° C., annealing for 30 seconds at 55°C., and extension for 30 seconds at 72° C. The fragment of theN-terminal region thus obtained was treated with restriction enzymes,BamHI and SalI and the fragment of the C-terminal region thus obtainedwas treated with restriction enzymes, Sail and XbaI. The fragments thustreated were cloned into the pDZ vector treated with restrictionenzymes, BamHI and XbaI, so as to construct a plasmidpDZ-1′NCgl2522(K/O).

The plasmid pDZ-1′NCgl2522(K/O) was transformed into Corynebacteriumglutamicum KCCM11138P and KCCM11240P of Reference Examples 1 and 2 byelectroporation, respectively so as to obtain transformants. Then, thetransformants were plated and cultured on BHIS plate (37 g/l of Braineheart infusion, 91 g/l of sorbitol, 2% agar) containing kanamycin (25μg/ml) and X-gal (5-bromo-4-chloro-3-indolin-D-galactoside) for colonyformation. From the colonies formed on the plate, blue-colored colonieswere selected as the strain introduced with the plasmidpDZ-1′NCgl2522(K/O).

The selected strains were cultured with shaking in CM medium (10 g/l ofglucose, 10 g/l of polypeptone, 5 g/l of yeast extract, 5 g/l of beefextract, 2.5 g/l of NaCl, 2 g/l of urea, pH 6.8) at 30° C. for 8 hours.Subsequently, each cell culture was serially diluted from 10⁻⁴ to 10⁻¹⁰.Then, the diluted samples were plated and cultured on anX-gal-containing solid medium for colony formation. From the coloniesformed, the white colonies which appear at relatively low frequency wereselected to finally obtain strains in which the gene encoding NCgl2522was deleted by secondary crossover. The strains finally selected weresubjected to PCR using a pair of primers of SEQ ID NO: 5 and 8 toconfirm deletion of the gene encoding NCgl2522. The Corynebacteriumglutamicum mutant strains were designated as KCCM11138P ΔNCgl2522 andKCCM11240P ΔNCgl2522, respectively.

<2-2> Preparation of NCgl2522-deleted Strain from ATCC13869-basedPutrescine-producing Strain

NCgl2522-deleted strain was prepared from Corynebacterium glutamicumATCC13869-based putrescine-producing strains, DAB12-a (argF deletion,NCgl1221 deletion, E. coli speC introduction, arg operon promotersubstitution; see Reference Example 1) and DAB12-b (argF deletion,NCgl1221 deletion, E. coli speC introduction, arg operon promotersubstitution, NCgl1469 deletion, see Reference Example 2) having thesame genotype as KCCM11138P and KCCM11240P which are Corynebacteriumglutamicum ATCC13032-based putrescine-producing strains.

Specifically, to examine the sequences of the gene encodingCorynebacterium glutamicum ATCC13869-derived NCgl2522 and the proteinexpressed therefrom, PCR was performed using the genomic DNA ofCorynebacterium glutamicum ATCC13869 as a template and a pair of primersof SEQ ID NOs: 5 and 8. At this time, PCR reaction was carried out for30 cycle of denaturation for 30 seconds at 95° C., annealing for 30seconds at 55° C., and extension for 2 minutes at 72° C. The PCR productthus obtained was separated by electrophoresis, and subjected tosequencing. As a result, it was found that the nucleotide sequence ofgene encoding Corynebacterium glutamicum ATCC13869-derived NCgl2522 isrepresented by SEQ ID NO: 22, and the amino acid sequence of proteinencoded thereby is represented by SEQ ID NO: 23. When the amino acidsequence of Corynebacterium glutamicum ATCC13032-derived NCgl2522 wascompared to that of the Corynebacterium glutamicum ATCC13869-derivedNCgl2522, they were found to have 98% sequence homology.

In order to delete the gene encoding Corynebacterium glutamicumATCC13869-derived NCgl2522, in the same manner as in Example <2-1>, PCRwas performed using the genomic DNA of Corynebacterium glutamicumATCC13869 as a template and two pairs of primers of Table 2 so as toamplify PCR fragments of the N-terminal and C-terminal regions ofNCgl2522 gene, respectively. These PCR fragments were electrophoresed toobtain the desired fragments. At this time, PCR reaction was carried outfor 30 cycle of denaturation for 30 seconds at 95° C., annealing for 30seconds at 55° C., and extension for 30 seconds at 72° C. The fragmentof the N-terminal region thus obtained was treated with restrictionenzymes, BamHI and SalI and the fragment of the C-terminal region thusobtained was treated with restriction enzymes, SalI and XbaL Thefragments thus treated were cloned into the pDZ vector treated withrestriction enzymes, BamHI and XbaI, so as to construct a plasmidpDZ-2′NCgl2522(K/O).

In the same manner as in Example <2-1>, the plasmid pDZ-2′NCgl2522(K/O)was transformed into Corynebacterium glutamicum DAB12-a and DAB12-b,respectively. Strains, in which the gene encoding NCgl2522 was deleted,were selected. Corynebacterium glutamicum mutant strains thus selectedwere designated as DAB12-a ΔNCgl2522 and DAB12-b ΔNCgl2522,respectively.

<2-3> Evaluation of Putrescine Productivity of NCgl2522-deleted Strain

In order to confirm the effect of NCgl2522 deletion on putrescineproductivity in the putrescine-producing strain, putrescineproductivities of the Corynebacterium glutamicum mutant strains preparedin Examples <2-1> and <2-2> were compared.

Specifically, 4 types of Corynebacterium glutamicum mutants (KCCM11138PΔNCgl2522, KCCM11240P ΔNCgl2522, DAB12-a ΔNCgl2522, and DAB12-bΔNCgl2522) and 4 types of parent strains (KCCM11138P, KCCM11240P,DAB12-a, and DAB12-b) were plated on CM plate media (1% glucose, 1%polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2%urea, 100 μl of 50% NaOH, 2% agar, pH 6.8, based on 1 L) containing 1 mMarginine, and cultured at 30° C. for 24 hours, respectively. 1 platinumloop of each strain thus cultured was inoculated in 25 ml of titermedium (8% Glucose, 0.25% soybean protein, 0.50% corn steep solids, 4%(NH₄)₂SO₄, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, 100 g of biotin, 3mg of thiamine hydrochloride, 3 mg of calcium-pantothenic acid, 3 mg ofnicotinamide, 5% CaCO₃, based on 1 L), and then cultured with shaking at30° C. and 200 rpm for 98 hours. 1 mM arginine was added to the mediafor culturing all strains. The putrescine concentration in each culturewas measured, and the results are shown in the following Table 3.

TABLE 3 Host Genotype Putrescine (g/L) KCCM11138P (−) 9.8 ΔNCgl2522 3.0KCCM11240P (−) 12.4 (KCCM11138P Δ NCgl1469) Δ NCgl2522 1.5 DAB12-a (−)10.2 Δ NCgl2522 0.7 DAB12-b (−) 13.1 (DAB12-a ^(Δ)NCgl1469) Δ NCgl25220.3

As shown in Table 3, a remarkable reduction in putrescine production wasobserved in 4 types of the NCgl2522-deleted Corynebacterium glutamicummutant strains.

EXAMPLE 3 Preparation of NCgl2522-enhanced Strain and Examination of itsPutrescine Productivity

<3-1> Introduction of NCgl2522 into Transposon Gene in ATCC13032Chromosome

In order to confirm high production of putrescine by additionalchromosomal insertion of NCgl2522 gene (containing a self promoterregion) in Corynebacterium sp. microorganism KCCM11138P havingputrescine productivity, NCgl2522 was introduced into a transposon gene.A vector for transformation, pDZTn (Korean Patent Publication No.10-2008-0033054) which allows introduction of the gene into a transposongene on the chromosome of Corynebacterium sp. microorganism was used.

The NCgl2522 gene containing the self promoter was amplified using thechromosome of ATCC13032 strain as a template and a pair of primers ofSEQ ID NO: 9 and 10 (see Table 4). At this time, PCR reaction wascarried out for 30 cycle of denaturation for 30 seconds at 95° C.,annealing for 30 seconds at 55° C., and extension for 30 seconds or 2minutes at 72° C. Through PCR, a gene fragment having a size of 1.88 kbwas obtained. This PCR product was electrophoresed in a 0.8% agarose gelto elute and purify a band of the desired size. pDZTn vector was treatedwith XhoI, and fusion cloning of the NCgl2522 PCR product of ATCC13032strain was performed. In-FusionHD Cloning Kit (Clontech) was used in thefusion cloning. The resulting plasmid was designated aspDZTn-1′NCgl2522.

TABLE 4 Primer Sequence (5′-3′) 1′NCgl2522-F-TTGTCGGGCCCACTAGTGGTGCGACTTCAATT- (SEQ ID NO: 9) GTGCTCTT NCgl2522-R-TGAATGAGTTCCTCGAGCTAGTGCG- (SEQ ID NO: 10) CATTATTGGCTCC

The plasmid pDZTn-1′NCgl2522 was transformed into Corynebacteriumglutamicum KCCM11138P described in Reference Example 1 byelectroporation to obtain transformants. From the transformants, astrain in which NCgl2522 was introduced into the transposon was selectedin the same manner as in Example 2.

PCR was performed using genomic DNA of the selected strain and a pair ofprimers of SEQ ID NOs: 9 and 10 to confirm that NCgl2522 was introducedinto the transposon by introduction of plasmid pDZTn-1′NCgl2522. At thistime, PCR reaction was carried out for 30 cycle of denaturation for 30seconds at 94° C., annealing for 30 seconds at 55° C., and extension for2 minutes at 72° C.

A Corynebacterium glutamicum mutant strain thus selected was designatedas KCCM11138P Tn: 1′NCgl2522.

<3-2> Preparation of NCgl2522 Promoter substituted Strain fromATCC13032-based Putrescine-Producing Strain

In order to enhance NCgl2522 activity in the putrescine-producingstrain, a CJ7 promoter (WO 2006/65095) was introduced in front of theNCgl2522 start codon on the chromosome.

First, a homologous recombination fragment containing a CJ7 promoterhaving the nucleotide sequence represented by SEQ ID NO: 24 and havingthe original NCgl2522 sequence at the both ends of the promoter wasobtained. Specifically, PCR was performed using the genomic DNA ofCorynebacterium glutamicum ATCC13032 as a template and a pair of primersof SEQ ID NOs: 11 and 12 to obtain the 5′-terminal region of CJ7promoter. At this time, PCR reaction was carried out for 30 cycle ofdenaturation for 30 seconds at 94° C., annealing for 30 seconds at 55°C., and extension for 30 seconds at 72° C. Further, PCR was performedusing a pair of primers of SEQ ID NOs: 13 and 14 under the sameconditions to obtain the CJ7 promoter region. Furthermore, PCR wasperformed using the genomic DNA of Corynebacterium glutamicum ATCC13032as a template and a pair of primers of SEQ ID NOs: 15 and 16 under thesame conditions to obtain the 3′-terminal region of CJ7 promoter. Theprimers used in promoter substitution are the same as in the followingTable 5.

TABLE 5 Primer Sequence (5′-3′) NCgl2522-L5 TGCAGGTCGACTCTAGAGTTCTGCG-(SEQ ID NO: 11) TAGCTGTGTGCC NCgl2522-L3 GGATCGTAACTGTAACGAATGG(SEQ ID NO: 12) CJ7-F CGTTACAGTTACGATCCAGAAACATCCCAGCGC- (SEQ ID NO: 13)TACTAATA CJ7-R AGTGTTTCCTTTCGTTGGGTACG (SEQ ID NO: 14) NCgl2522-R5CAACGAAAGGAAACACTATGACTTCAGAAAC- (SEQ ID NO: 15) CTTACAGGCG NCgl2522-R3TCGGTACCCGGGGATCCCACAAAAAGCG- (SEQ ID NO: 16) TAGCGATCAACG

Each PCR product thus obtained was fusion-cloned into pDZ vector treatedwith BamHI and XbaI. In-FusionHD Cloning Kit (Clontech) was used in thefusion cloning. The resulting plasmid was designated aspDZ-P(CJ7)-1′NCgl2522.

The plasmid pDZ-P(CJ7)-1′NCgl2522 thus prepared was transformed intoCorynebacterium glutamicum KCCM11138P and KCCM11240P according toReference Examples 1 and 2 by electroporation so as to preparetransformants. The transformants thus prepared were inoculated in CMmedia and cultured with shaking at 30° C. for 8 hours. Each cell cultureobtained therefrom was diluted from 10⁻⁴ to 10⁻¹⁰, and plated andcultured on BHIS plate containing 25 μg/ml of kanamycin and X-gal forcolony formation.

The white colonies appear at relatively low frequency, compared tomajority of the colonies having blue color, and were selected to finallyobtain a strain in which the NCgl2522 promoter was substituted with theCJ7 promoter by secondary crossover. PCR was performed using the genomicDNA of the selected strain as a template and a pair of primers of SEQ IDNOs: 13 and 16 to confirm that the CJ7 promoter was introduced in frontof the NCgl2522 start codon on the chromosome by introduction of theplasmid pDZ-1′CJ7(NCgl2522). At this time, PCR reaction was carried outfor 30 cycle of denaturation for 30 seconds at 94° C., annealing for 30seconds at 55° C., and extension for 1 minute at 72° C.

Corynebacterium glutamicum mutant strains thus selected were designatedas KCCM11138P P(CJ7)-NCgl2522 and KCCM11240P P(CJ7)-NCgl2522,respectively.

<3-3> Introduction of NCgl2522 Gene into Transposon Gene on ATCC13869Chromosome

In order to confirm high production of putrescine by additionalchromosomal insertion of NCgl2522 gene in Corynebacterium glutamicumATCC13869-derived putrescine strain, introduction of NCgl2522(containing the promoter region) into a transposon gene was determinedNCgl2522 gene was amplified using the chromosome of ATCC13869 strain asa template and a pair of primers of SEQ ID NOs: 17 and 10 (see Table 6).At this time, PCR reaction was carried out for 30 cycle of denaturationfor 30 seconds at 94° C., annealing for 30 seconds at 55° C., andextension for 30 seconds or 2 minutes at 72° C. Through PCR, a genefragment having a size of 1.97 kb was obtained. The NCgl2522 PCRfragment thus prepared was fusion-cloned into pDZTn vector treated withXhoI. In-FusionHD Cloning Kit (Clontech) was used in the fusion cloning.The resulting plasmid was designated as pDZTn-2′NCgl2522.

TABLE 6 Primer Sequence (5′-3′) 2′NCgl2522-F-TTGTCGGGCCCACTAGTCTTCAATTCGAGTT- (SEQ ID NO: 17) GCTGCCAC NCgl2522-R-TGAATGAGTTCCTCGAGCTAGTGCG- (SEQ ID NO: 10) CATTATTGGCTCC

The plasmid pDZTn-2′NCgl2522 was transformed into Corynebacteriumglutamicum DAB12-a in the same manner as in Example <3-1> to confirmintroduction of NCgl2522 into the transposon.

A Corynebacterium glutamicum mutant strain thus selected was designatedas DAB12-a Tn:2′NCgl2522.

<3-4> Preparation of NCgl2522 Promoter-substituted Strain fromATCC13869-based Putrescine-Producing Strain

In order to introduce the CJ7 promoter in front of the NCgl2522 startcodon of Corynebacterium glutamicum ATCC13869, PCR was performed usingthe genomic DNA of Corynebacterium glutamicum ATCC13869 as a templateand three pairs of primers given in the following Table 7 in the samemanner as in Example <3-2>, respectively. Consequently, PCR fragments ofthe CJ7 promoter region, its N-terminal region and C-terminal regionwere amplified and then electrophoresed to obtain the desired fragments.At this time, PCR reaction was carried out for 30 cycle of denaturationfor 30 seconds at 94° C., annealing for 30 seconds at 55° C., andextension for 30 seconds at 72° C. PCR fragments of the CJ7 promoterregion, its N-terminal region and C-terminal region thus obtained werefusion-cloned into pDZ vector treated with BamHI and XbaI. In-FusionHDCloning Kit (Clontech) was used in the fusion cloning. The resultingplasmid was designated as pDZ-P(CJ7)-2′NCgl2522.

TABLE 7 Primer Sequence (5′-3 ′) 2′NCgl2522-L5TGCAGGTCGACTCTAGACAATTCGAGTT- (SEQ ID NO: 18) GCTGCCACAC NCgl2522-L3GGATCGTAACTGTAACGAATGG (SEQ ID NO: 12) CJ7-FCGTTACAGTTACGATCCAGAAACATCCCAGCGC- (SEQ ID NO: 13) TACTAATA CJ7-RAGTGTTTCCTTTCGTTGGGTACG (SEQ ID NO: 14) NCgl2522-R5CAACGAAAGGAAACACTATGAT- (SEQ ID NO: 19) TTCAGAAACTTTGCAGGCG NCgl2522-R3TCGGTACCCGGGGATCCCACAAAAAGCG- (SEQ ID NO: 17) TAGCGATCAACG

The plasmid pDZ-′P(CJ7)-2′NCgl2522 was transformed into each ofCorynebacterium glutamicum DAB12-a and DAB12-b in the same manner as inExample <3-2> to select strains, in which the CJ7 promoter wasintroduced in front of the NCgl2522 start codon. Corynebacteriumglutamicum mutant strains thus selected were designated as DAB12-aP(CJ7)-NCgl2522 and DAB12-b P(CJ7)-NCgl2522.

<3-5> Evaluation of Putrescine Productivity of NCgl2522-enhanced Strain

In order to confirm the effect of NCgl2522 activity enhancement bypromoter substitution on putrescine productivity in theputrescine-producing strain, putrescine productivities of 6 types ofCorynebacterium glutamicum mutant strains (KCCM11138P Tn: 1′NCgl2522,KCCM11138P P(CJ7)-NCgl2522, KCCM11240P P(CJ7)-NCgl2522, DAB12-aTn:2′NCgl2522, DAB12-a P(CJ7)-NCgl2522 and DAB12-b P(CJ7)-NCgl2522)prepared in Examples <3-1> to <3-4> and 4 types of parent strains(KCCM11138P, KCCM11240P, DAB12-a and DAB12-b) were compared. Each strainwas cultured in the same manner as in Example 2-3, and the putrescineconcentration in each culture was measured, and the results are shown inthe following Table 8.

TABLE 8 Host Genotype Putrescine (g/L) KCCM11138P (−) 9.8 Tn: 1′NCgl252211.7 P(CJ7)-NCgl2522 13.5 KCCM11240P (−) 12.4 P(CJ7)-NCgl2522 15.5DAB12-a (−) 10.2 Tn: 2′NCgl2522 12.3 P(CJ7)-NCgl2522 14.1 DAB12-b (−)13.1 P(CJ7)-NCgl2522 15.9

As shown in Table 8, an increase in putrescine production was observedin all 6 types of Corynebacterium glutamicum mutant strains in whichNCgl2522 activity was enhanced by additional introduction of NCgl2522into the transposon or by promoter substitution.

EXAMPLE 4 Measurement of Intracellular Putrescine Concentration ofNCgl12522-enhanced Strain

In order to confirm that intracellular putrescine concentration isreduced by an enhancing ability to export putrescine in theCorynebacterium glutamicum mutant strain having enhanced NCgl2522activity, intracellular putrescine concentrations in Corynebacteriumglutamicum mutant strain KCCM11138P Tn: 1′NCgl2522 and in parent strainKCCM11138P were measured by extraction using an organic solvent.Intracellular metabolite analysis was carried out in accordance with amethod described in the literature (Nakamura J et al., Appl. Environ.Microbiol. 73(14): 4491-4498, 2007).

First, Corynebacterium glutamicum mutant strain KCCM11138P Tn:1′NCgl2522and parent strain KCCM11138P were inoculated in 25 ml of CM liquid media(1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract,0.25% NaCl, 0.2% urea, 100 l of 50% NaOH, pH 6.8, based on 1 L)containing 1 mM arginine, and cultured with shaking at 30° C. and 200rpm. When cell growth reached exponential phase during cultivation,cells were isolated from the culture media by rapid vacuum filtration(Durapore HV, 0.45 m; Millipore, Billerica, Mass.). The cell-adsorbedfilter was washed with 10 ml of cooled water twice, and then dipped inmethanol containing 5 M morpholine ethanesulfonic acid and 5 Mmethionine sulfone for 10 minutes.

The extraction liquid obtained therefrom was mixed well with an equalvolume of chloroform and 0.4-fold volume of water, and the aqueous phasewas only applied to a spin column to remove protein contaminants. Thefiltered extraction liquid was analyzed by capillary electrophoresismass spectrometry, and the results are shown in the following Table 9.

TABLE 9 Strain Putrescine (mM) KCCM11138P 7 KCCM11138P Tn: 1′NCgl2522 2

As shown in Table 9, a reduction in the intracellular putrescineconcentration was observed in Corynebacterium glutamicum mutant strainKCCM11138P Tn:1′NCgl2522 having enhanced NCgl2522 activity, compared toparent strain KCCM11138P, It suggests that an improved ability to exportputrescine by enhancement of NCgl2522 activity in Corynebacteriumglutamicum mutant strain KCCM11138P Tn: 1′NCgl2522, leads to effectiveextracellular export of intracellular putrescine.

EXAMPLE 5 Evaluation of Putrescine Resistance of NCgl2522-deleted or-enhanced Strain

In order to examine the effect of NCgl2522 on putrescine resistance,putrescine resistance of KCCM11240P, KCCM11240P ΔNCgl2522, andKCCM11240P P(CJ7)NCgl2522 strains was evaluated.

Each strain was inoculated in 2 ml of CM liquid containing 1 mM argininemedium and cultured at 30° C. for about 10 hours, followed by dilutionin this order of 10⁵, 10⁴, 10³, 10² and 10¹. Each dilution thus preparedwas spotted on 0 M or 0.8 M putrescine-containing CMA plate (1% glucose,1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2%urea, 1.8% agar, 1 mM arginine, pH 6.8, based on 1 L) and then culturedat 30° C. for 48 hours to compare growth differences between strains.

As a result, the strains showed two different growth patterns. As shownin FIG. 2, the NCgl2522 gene-deleted strain did not grow under theconditions of a high concentration of putrescine, whereas the NCgl2522gene-enhanced strain grew up under the same conditions. This resultshows that the increased cell growth of KCCM11240P P(CJ7)-NCgl2522 underthe conditions of a high concentration of putrescine compared to theparent strain is caused by the increased ability to export putrescinedue to enhancement of NCgl2522 gene. As a result, introduction andenhancement of NCgl2522 are essential for fermentation ofhigh-concentrations of putrescine.

EXAMPLE 6 Putrescine Fermentation Through Introduction of NCgl2522 intoE. coli

In order to confirm whether putrescine production is increased whenNCgl2522 of Corynebacterium glutamicum ATCC13032 is expressed in thewild-type E. coli strain W3110 having a putrescine biosynthetic pathway,a vector expressing speC which is a putrescine synthetic enzyme or avector expressing NCgl2522 were introduced into W3110.

In order to prepare the speC-expressing vector, W3110 chromosome as atemplate and a pair of primers of SEQ ID NOs: 34 and 35 were used toamplify a speC gene fragment of about 2.1 kb (see Table 10). This PCRproduct was electrophoresed in a 0.8% agarose gel, and then a band ofthe desired size was eluted and purified. pSE280 vector (Invitrogen)containing Trc promoter was treated with NcoI and EcoRI, and then thespeC PCR product was fusion-cloned into this vector. In-Fusion® HDCloning Kit (Clontech) was used in the fusion cloning. The resultingplasmid was designated as pSE280-speC.

In order to prepare the NCgl2522-expressing vector, pSE280 as a templateand a pair of primers of SEQ ID NOs: 36 and 37 were used to obtain a Trcpromoter fragment, and Corynebacterium glutamicum ATCC13032 chromosomeas a template and a pair of primers of SEQ ID NOs: 38 and 39 were usedto obtain an NCgl2522 fragment. These PCR products were electrophoresedin a 0.8% agarose gel, and then bands of the desired size were elutedand purified. The trc promoter fragment and the NCgl2522 fragment werefusion-cloned into pcc1BAC treated with HindIII. The resulting plasmidwas designated as pcc1BAC-P(trc)NCgl2522.

TABLE 10 Primer Sequence (5′-3 ′) SPEC-F CACAGGAAACAGAC- (SEQ ID NO: 34)CATGGATGAAATCAATGAATATTGCCGCCA SPEC-R GTG- (SEQ ID NO: 35)CAGGTGCTGAATTCTTACTTCAACACATAAC- CGTACAAC Ptrc-FTGCAGGCATGCAAGCTTCGACATCATAAC- (SEQ ID NO: 36) GGTTCTGGC Ptrc-RATTATACGAGCCGGATGATTAATTG (SEQ ID NO: 37) NCgl2522-FCATCCGGCTCGTATAATATGACTTCAGAAAC- (SEQ ID NO: 38) CTTACAGGC NCgl2522-RATAGAATACTCAAGCTTCTAGTGCG- (SEQ ID NO: 39) CATTATTGGCTCC

The plasmids, pSE280-speC or pcc1BAC-P(trc)-NCgl2522, were transformedinto W3110. Transformation into E. coli was carried out using 2×TSSsolution (Epicentre). pSE280-speC-introduced E. coli was plated andcultured on an ampicillin (100 μg/ml) containing LB plate (10 g ofTryptone, 5 g of yeast extract, 10 g of Nacl, 2% agar, based on 1 l) forcolony formation. pcc1BAC-P(trc)-NCgl2522-introduced E. coli was platedand cultured on a chloramphenicol (35 μg/ml)-containing LB plate forcolony formation. Putrescine productivities of the strains thus obtainedwere examined.

Specifically, W3110, W3110 pSE280-speC, and W3110pcc1BAC-P(trc)-NCgl2522 were inoculated on LB, LA and LC plates,respectively and cultured at 37° C. for 24 hours, and then inoculated in25 ml of titer medium (2 g of (NH₄)₂PO₄, 6.75 g of KH₂PO₄, 0.85 g ofcitric acid, 0.7 g of MgSO₄.7H₂O, 0.5% (v/v) trace element, 10 g ofglucose, 3 g of AMS, 30 g of CaCO₃, based on 1 L) and cultured at 37° C.for 24 hours. A trace metal solution contains 5 M HCl, 10 g ofFeSO₄.7H₂O, 2.25 g of ZnSO₄.7H₂O, 1 g of CuSO₄.5H₂O, 0.5 g ofMnSO₄.5H₂O, 0.23 g of Na₂B₄O₇.10H₂O, 2 g of CaCl₂.2H₂O, and 0.1 g of(NH₄)₆Mo₇O₂.4H₂O in 1 L.

The putrescine concentration in each culture was measured, and theresults are shown in the following Table 11.

TABLE 11 Host Plasmid Putrescine (mg/L) W3110 (−) 11 pSE280-speC 56pcc1BAC-P(trc)-NCgl2522 250

As shown in Table 11, high putrescine production was observed in theW3110 pcc1BAC-P(trc)-NCgl2522 strain introduced with NCgl2522, comparedto W3110pcc1BACpSE280-speC strain introduced with the putrescinebiosynthetic enzyme, speC.

This result demonstrates that NCgl2522 protein also has the ability toexport putrescine in E. coli.

The present inventors found that additional introduction of NCgl2522into the transposon of Corynebacterium sp. microorganism KCCM11138Phaving putrescine productivity was performed to enhance NCgl2522activity of Corynebacterium glutamicum strain, and thus putrescine couldbe produced in a high yield owing to the increased ability to exportputrescine, and they designated the strain as Corynebacterium glutamicumCC01-0510, and deposited under the Budapest Treaty to the Korean CultureCenter of Microorganisms (KCCM) on Mar. 8, 2013, with Accession No.KCCM11401P.

Based on the above description, it should be understood by those skilledin the art that other specific embodiments may be employed in practicingthe invention without departing from the technical idea or essentialfeatures of the invention. In this regard, the above-described examplesare for illustrative purposes only, and the invention is not intended tobe limited by these examples. The scope of the present invention shouldbe understood to include all of the modifications or modified formderived from the meaning and scope of the following claims or itsequivalent concepts, rather than the above detailed description.

EFFECT OF THE INVENTION

A Corynebacterium sp. microorganism having improved putrescineproductivity of the present invention is modified to have enhancedNCgl2522 activity of exporting intracellular putrescine, compared to itsendogenous activity, resulting in increased extracellular export ofputrescine and increased putrescine resistance.

Further, when NCgl2522 was expressed in E. coli containing a putrescinesynthetic pathway of the present invention, the amount of extracellularputrescine was found to increase. Accordingly, Corynebacteriumglutamicum-derived NCgl2522 can be applied to a microorganism havingputrescine productivity, which can be widely used in the effectiveproduction of putrescine.

What is claimed is:
 1. A recombinant microorganism having enhancedputrescine productivity, wherein the recombinant microorganism comprisesa modification that enhances expression of a polynucleotide encoding aprotein comprising the amino acid sequence of SEQ ID NO: 21 or 23 ascompared to the expression in the same microorganism without themodification that enhances expression, wherein the microorganism furthercomprises a modification that enhances expression of a polynucleotideencoding an ornithine decarboxylase (ODC) as compared to the expressionin the same microorganism without the modification that enhancesexpression, wherein the modification that enhances expression istransformation with an expression vector comprising the polynucleotideor replacing the promoter of the polynucleotide with a heterologouspromoter.
 2. The recombinant microorganism of claim 1, wherein therecombinant microorganism further comprises a modification that reducesexpression of a gene encoding an ornithine carbamoyltransferase (ArgF)and a modification reduces expression of a gene encoding a proteininvolved in glutamate export as compared to the expression in the samemicroorganism without the modifications that reduces expression, whereinthe modification that reduces expression is inactivation or disruptionof the gene.
 3. The recombinant microorganism of claim 2, wherein theornithine carbamoyltransferase (ArgF) comprises the amino acid sequenceof SEQ ID NO: 29, the protein involved in glutamate export comprises theamino acid sequence of SEQ ID NO: 30, and the ornithine decarboxylase(ODC) comprises the amino acid sequence of SEQ ID NO:
 33. 4. Therecombinant microorganism of claim 1, wherein the recombinantmicroorganism further comprises a modification that enhances expressionof a polynucleotide encoding an acetyl-gamma-glutamyl-phosphatereductase (ArgC), a polynucleotide encoding an acetylglutamate synthaseor ornithine acetyltransferase (ArgJ), a polynucleotide encoding anacetylglutamate kinase (ArgB), and a polynucleotide encoding anacetylornithine aminotransferase (ArgD), as compared to the expressionin the same microorganism without the modification that enhancesexpression, wherein the modification that enhances expression istransformation with an expression vector comprising the polynucleotideor replacing the promoter of the polynucleotide with a heterologouspromoter.
 5. The recombinant microorganism of claim 4, wherein theacetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamatesynthase or ornithine acetyltransferase (ArgJ), acetyl glutamate kinase(ArgB), and acetylornithine aminotransferase (ArgD) comprise the aminoacid sequences of SEQ ID NOs: 25, 26, 27 and 28, respectively.
 6. Therecombinant microorganism of claim 1, wherein the recombinantmicroorganism further comprises a modification that reduces expressionof a gene encoding an acetyltransferase as compared to the activity inthe same microorganism without the modification that reduces expression,wherein the modification that reduces expression is inactivation ordisruption of the gene.
 7. The recombinant microorganism of claim 6,wherein the acetyltransferase comprises the amino acid sequence of SEQID NO: 31 or
 32. 8. The recombinant microorganism of claim 1, whereinthe microorganism is an Escherichia sp. or a Corynebacterium sp.
 9. Therecombinant microorganism of claim 8, wherein the microorganism is E.coli or Corynebacterium glutamicum.
 10. A method for producingputrescine, comprising the steps of: (i) culturing the recombinantmicroorganism of any one of claims 1 to 9 to obtain a cell culture; and(ii) recovering putrescine from the cultured recombinant microorganismor the cell culture.